The uterine environment, which, if hostile/non-receptive, can be responsible for poor implantation rates of good quality embryos in human and animals alike. It is believed that an inadequately primed uterine environment may also be responsible for many cases of reproductive failure in terms of failed implantation and spontaneous abortion. Similarly, a failure in uterine priming is recognised in humans as being causative to pregnancy complications such as pre-eclampsia and foetal growth restriction by preventing appropriate placental development.
Genetically altered or modified animals provide valuable models for testing novel gene and drug therapies in vivo and are the main reason the numbers of animal experiments have been rising in the last decade. In the UK, over four times as many scientific procedures using genetically modified animals were carried out in 2011 as compared to 1995. The use of genetically modified animals now represents over 50% of all scientific procedures on animals. The largest category of use is breeding (to produce genetically modified animals), with rodents accounting for almost 1.8 million procedures in the UK in 2012 alone, on a background trend for this number to increase annually. Embryo transfer in rodents underpins the development of transgenic approaches, re-derivation of specific strains and facilitates the transport of animal-lines across large distances. Typically, embryo transfer requires induction of pseudopregnancy in recipient females. This phenomenon prepares the uterus for implanting embryos, however, the success rate of transferring genetically modified embryos, despite induction of pseudopregnancy, remains relatively low.
Mice are spontaneous ovulators and can become pseudopregnant following an estrus in which the female is mated with a genetically sterile male such as the T145H-Re strain (which is sterile due to a chromosomal translocation) obtainable from Harlan Laboratories Inc or a vasectomised male. Both sets of males ejaculate seminal plasma devoid of functional sperm. However, both genetically sterile and vasectomised mice are relatively costly. In the instance of vasectomised males, sterility cannot be guaranteed to be 100% effective and needs testing for each male, while the production of genetically sterile males generates unwanted surplus females.
Alternatively, pseudopregnancy can be induced by simulating the normal vaginal stimuli attained by mating with artificial mechanical stimulation, for example by a vibrating engraving tool (Kenney et al; J Reprod. Fert. 1977, 49, 305-309). It was found that the number and rate of intromissions were crucial influences on reproductive success (Diamond; Science, 1970, 169, 4, 995-997). Whilst this approach has seen some success in rats and mice mechanical stimulation had no effect on the induction of pseudopregnancy in the Golden Hamster (Diamond et al J. Reprod. Fert. 1968, 17, 165-168). When the female is mated with an infertile male or mechanically stimulated, the corpus luteum persists without an embryo, leading to pseudopregnancy. The female will develop mammary glands, lactate and build nests in the pseudopregnant state. There is a need to improve the methods of inducing a pseudopregnant state in laboratory test animals.
Although the protocols for embryo transfer in an array of rodent species are relatively well-established, their poor optimization means that there is a significant wastage of animals, raising a number of financial and ethical issues in animal units worldwide. The prior art standard approach currently relies on mating recipient females with vasectomised males to induce pseudopregnancy rather than mechanical stimulation, where copulatory activity and seminal exposure of the maternal reproductive tract triggers a neuroendocrine and localised (to the uterus, principally) inflammatory response involving a complex cascade of cytokine and prostaglandin-mediated events geared towards creating an immunopermissive environment in the uterus, thereby favouring pre-implantation embryo development and/or blastocyst implantation and the establishment of pregnancy. Even in the absence of fertilisation, luteal development and progesterone production are supported, and the maternal physiology is orchestrated to render the uterus receptive to transferred embryos for up to 10-13 days. This technique is routinely used to support the development of normal (cryopreserved strain regeneration/re-derivation) or genetically modified (transgenic/chimaeric/cloned) embryos.
However, the efficacy of this approach is limited. Typically, four times as many females are prepared for the procedure compared to those becoming pregnant. When implanting fresh or frozen embryos this represents a considerable wastage of valuable biological material and effort. Moreover, numbers of young vasectomised males also need to be maintained alongside the prospective recipients: these can only mate 2-3 times a week and are typically replaced every 6-9 months in order to maintain performance.
Mating predominantly occurs when the recipient female is in estrus. The estrus cycle lasts 4-5 days in the mouse and rat (equivalent to a woman's average 28 day menstrual cycle), which leads to the need to rely on a large pool of potential recipient females to take part in potential matings with vasectomised or otherwise sterile males. Typically, 75% of recipients are not in estrus in randomly cycling populations, leading to large numbers of females and vasectomised or otherwise sterile males being kept and, in the case of the former, often not used as surrogates in order to guarantee adequate numbers of recipients for use in timed transfers. This is particularly evident in instances where the embryos to be transferred are particularly valuable. Improvements to this approach have relied on timed estrus induction via the Whitten effect in recipient females. This strategy relies on pheremonal stimulation of recipient females, which typically brings them into estrus 3 days after exposure to stud male urine-soiled bedding. However, the cycling stage of females at the time of pheremonal exposure, proximity to stud cages and the age of recipients can all have adverse effects on the reliability of this approach, making it relatively inefficient.
The chances of females being in estrus (sexually receptive) at the right time is 1:4 due to the length of their cycle (4 days). Thus, if 4 recipients are required, 16 females will be mated to 16 males, which translates to a 25% success rate. This figure can be even lower as some females will refuse to mate with their partner. The key point is that although most breeders either select females in estrus, or induce estrus before mating with sterile males, still only a relatively low percentage (often about 50%—but as low as 15% in some facilities) of oestrus females will become ‘plugged’ and so assumed to be pseudopregnant. Furthermore, females also have a very limited functional lifespan of a few months of age as embryo transfer recipients. Females rapidly accumulate abdominal fat as they mature, making laparotomic embryo transfers (the most common and successful method) technically too challenging.
It is known that seminal fluid ejaculate is a complex mixture rich a variety of cytokines and prostaglandins, and that some of the cytokines may have a positive effect on the vaginal/uterine environment and others may have deleterious effects which both and ultimately affect receptivity to embryo implantation. For example, the pro-inflammatory cytokine GM-CSF (CSF-2) is present in seminal fluid ejaculate and is also known to be released by endometrial epithelial cells in response to seminal plasma within hours of coitus (Robertson et al. Reprod Fertil Dev, 1990. 2(4): p. 359-68) and that maternal GM-CSF is required for foetal viability and growth (Robertson et al. Biology of Reproduction Feb. 1, 1999 vol. 60 no. 2 251-261). Furthermore WO1999067364 A1 discloses media supplemented with GM-CSF to promote blastocyst growth and WO 2014087218 describes GM-CSF as sole acting substance to prevent recurrent miscarriage.
The cytokine IL-12 is also present in seminal fluid ejaculate and has been shown to be deleterious to pregnancy (Reina et al. Am J Reprod Immunol. 2004 May; 51(5):345-51) and to be abortifacient in high concentrations, particularly in synergy with IL-18 and IL-2, via activation of maternal lymphocytes (Hayakawa et al. Am J Reprod Immunol. 1999; 41: 320-329). Other studies have shown that IL-12 injections (100 ng i.p. daily on days 5, 6, 7 and 8 of pregnancy) into CBA/J mice caused increased abortion rate when compared to controls (Zenclussen et al Scand J Immunol. 2002 June; 55(6):560-9).
By the compositions and methods of the present invention it is envisaged that the need for vasectomised or otherwise sterile male mice can be dramatically reduced along with a significant reduction of female mice usage.
The present invention aims to improve the pregnancy rates in mammalian females in terms of established pregnancies and/or increased litter number following artificial or natural insemination or following transplantation of fresh or frozen or otherwise preserved embryos.